Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana, Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana, rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its applications in the future.

中文翻译：

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CRISPR-Cas9 系统在拟南芥、水稻和甘氨酸最大基因组中的靶标特异性

近年来，成簇的规则间隔短回文重复序列 (CRISPR) 是细菌中的一类免疫相关序列，已被开发为编辑不同细胞和生物体中真核基因组的强大工具。CRISPR-Cas9 系统可以识别与原始间隔区相邻基序位点相邻的上游 20 个核苷酸（引导序列），并触发双链 DNA 切割以及 DNA 修复机制，最终导致敲除、敲入或位点特异性诱变。然而，由引导序列错误识别引起的脱靶效应是其主要缺点并限制了其广泛应用。在这项研究中，对拟南芥、水稻（水稻）和最大甘氨酸中所有指导序列的特异性进行了全局分析(大豆)进行。结果，建立了一个简单的管道和三个全基因组数据库，并为科学界共享。对于 CRISPR-Cas9 的每个靶位点，计算和评估特异性评分和脱靶数。拟南芥、水稻和大豆的脱靶数平均值分别为 27.5、57.3 和 174.7。这些植物之间的比较分析表明，脱靶效应的频率与基因组大小、染色体位点、基因密度和鸟嘌呤胞嘧啶 (GC) 含量相关。我们的研究结果有助于更好地了解植物中的 CRISPR-Cas9 系统，并将有助于在未来的应用中最大限度地减少脱靶效应。